There is an increasing concern in the medical community regarding the emergence of strains of bacteria and other pathogens that are resistant to known antibiotics. Scientists, therefore, are attempting to discover new therapies that are effective through novel modes of action.
The topoisomerase family of enzymes is a target for several important anti-tumor and anti-infective drugs (A. Kornberg and T. Baker. 1991. DNA Replication (W. H. Freeman and Company, New York); Y. Pommier, P. Pourquier, Y. Fan and D. Strumberg. 1998. Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim. Biophys. Acta 1400, 83-106). Topoisomerases are ubiquitous and essential enzymes in cells. By introducing transient breaks into the helix, topoisomerases relax the DNA superhelical tension that arises in cells as a result of several nuclear processes including DNA replication, transcription, chromatin assembly, recombination, and chromosome segregation. The breaks that they generate may be single or double-stranded breaks, depending on the specific type of topoisomerase.
Compounds that act as effective cellular inhibitors of topoisomerases are expected to act as cytotoxic agents through the disruption of the normal cell division process. Such agents that are sufficiently potent and selective will be of great use as antibacterial and antifungal agents. Topoisomerases are also encoded by the genetic material in certain viruses, so development of topoisomerase inhibitors that are effective against viral topoisomerases may provide effective antiviral agents. Further, because cell division is an important characteristic of cancers and other proliferative diseases, agents that inhibit topoisomerases will also be useful as antineoplastic agents.
Many inhibitors trap the covalent topoisomerase-DNA intermediate, leading to the persistence of DNA breaks that disrupt cell growth. For example, amptothecin traps covalent intermediates made by human topoisomerase IB and thereby kills rapidly dividing cancer cells. The anti-Leishmania effect of pentavalent antimonials has been proposed to be a result of inhibiting the parasitic topoisomerase IB (A. K. Chakraborty and H. K. Majumder. 1988. Mode of action of pentavalent antimonials: specific inhibition of type I DNA topoisomerase of Leishmania donovani. Biochem. Biophys. Res. Commun. 152, 605-611). The marine natural product sansalvamide A1 was recently found to inhibit the topoisomerase of the pathogenic poxvirus molluscum contagiosum (MCV) (Y. Hwang, D. Rowley, D. Rhodes, J. Gertsch, W. Fenical and F. D. Bushman. 1999. Mechanism of inhibition of a poxvirus topoisomerase by the marine natural product sansalvamide A. Mol. Pharmacol. 55, 1049-1053). Emphasizing the toxicity of trapped covalent intermediates is the recent discovery of a DNA repair pathway dedicated solely to removing stalled intermediates from cellular DNA (J. J. Pouliot, K. C. Yao, C. A. Robertson and H. A. Nash. 1999. Yeast Gene for a Tyr-DNA Phosphodiesterase that Repairs Topoisomerase I Complexes. Science 286, 552-555).
Although previous methods for detecting topoisomerase inhibitors have been available, (see, e.g., Andrea et al., (1991) Mol. Pharmacol. 40(4):495-501; Muller et al., Nucleic Acids Res. 17(22):9499; Lynch et al., U.S. Pat. No. 5,998,152), the search for effective inhibitors for topoisomerases has been difficult due to the fact that a good screening assay does not exist. Previously available assays were not amenable to high throughput screening methods such as are needed to screen large libraries or groups of potential inhibitors. The known gel-based assays for topoisomerase function are not convenient for monitoring large numbers of tests. Other previously available assays required multiple steps or reagents. Prior assays required multiple steps that often identified compounds with non-specific characteristics, which often required further evaluation to determine its activity, e.g., whether the compound inhibited formation or trapping of covalent complexes. In addition, previously available assays employed reagents that often tainted or distorted the results of the assay or compound. For example, denaturants were often employed that disrupted enzymatic activity of the topoisomerase. Also, many of the currently available assays require the use of radioactive compounds and/or suffer from a lack of sensitivity. Thus, there remains a continuing need for high-throughput screening assays for inhibitors active against topoisomerases. There also remains a need for a method of treating infectious diseases and cancers by administering a topoisomerase inhibitor.
The present invention provides high-throughput methods of screening compounds capable of modulating topoisomerase activity by incubating at least a first nucleic acid, a topoisomerase and a potential topoisomerase-modulating compound, wherein the nucleic acid is operatively associated with at least one tag, and assaying for nucleic acid religation. It is then possible to measure the level of substrate nucleic acid religation activity in the presence and absence of the topoisomerase-modulating compound, wherein the level of religation activity is inversely proportional to the effectiveness of the topoisomerase-inhibitory compound. The nucleic acid may be single-stranded or double-stranded DNA, or single-stranded or double-stranded RNA. The tag may be a detection tag or an affinity tag. The method may involve incubating at least a first nucleic acid and a second nucleic acid, and the first nucleic acid may be operatively associated with an affinity tag, and the second nucleic acid may be operatively associated with a detection tag. The topoisomerase-modulating compound may be a topoisomerase inhibitor or an activator. The topoisomerase may be a Type I, Type II, Type III or Type IV topoisomerase. The screening assay may be performed on a solid support or in a liquid phase. The nucleic acid and topoisomerase may be covalently complexed, wherein the topoisomerase retains its religation activity.
The present invention also provides methods of treating cancer or an infection by a pathogen by administering a pharmaceutical composition including a topoisomerase inhibitor to a patient in need thereof. The pathogen may be a virus, bacterium, fungus or parasite.
The present invention further provides a kit for screening compounds that modulate topoisomerase religation activity that contains (a) a substrate nucleic acid having a first tag, (b) a religation nucleic acid having a second tag, (c) a topoisomerase, and (d) a means for measuring nucleic acid religation activity of a test mixture including (a), (b) and (c) in the presence or absence of a topoisomerase-modulating compound.
The present invention also provides a high-throughput method of screening compounds capable of modulating nucleic acid-modifying enzymatic activity by incubating at least a first nucleic acid, a nucleic acid-modifying enzyme and a potential enzyme-modulating compound, wherein the nucleic acid has at least one tag, and assaying for nucleic acid religating or cleavage.